Use of suspensions of gas bubbles in a carrier liquid, as efficient ultrasound reflectors is well known in the art. The development of these suspensions as means for enhancement of ultrasound imaging followed early observations that rapid intravenous injections of aqueous solutions can cause dissolved gases to come out of solution by forming bubbles. Due to their substantial difference in acoustic impedance relative to blood, these intravascular gas bubbles were found to be excellent reflectors of ultrasound. The injection of suspensions of gas bubbles in a carrier liquid into the blood stream of a living organism strongly reinforces ultrasonic echography imaging, thus enhancing the visualization of internal organs. Since imaging of organs and deep seated tissues can be crucial in establishing medical diagnosis, a lot of effort has been devoted to the development of stable suspensions of highly concentrated gas bubbles which at the same time would be simple to prepare and administer, would contain a minimum of inactive species and would be capable of long storage and simple distribution.
The simple dispersion of free gas bubbles in an aqueous medium is however of limited practical interest, since these bubbles are in general not stable enough to be useful as ultrasound contrast agents.
Interest has accordingly been shown in methods of stabilizing gas bubbles for echography and other ultrasonic studies, for example using emulsifiers, oils, thickeners or sugars, or by entraining or encapsulating the gas or a precursor thereof in a variety of systems. These stabilized gas bubbles are generally referred to in the art as “microvesicles”, and may be divided into two main categories. A first category of stabilized bubbles or microvesicles is generally referred to in the art as “microbubbles” and includes aqueous suspensions in which the bubbles of gas are bounded at the gas/liquid interface by a very thin envelope involving a surfactant (i.e. an amphiphilic material). A second category of microvesicles is generally referred to in the art as “microballoons” or “microcapsules” and includes suspensions in which the bubbles of gas are surrounded by a solid material envelope formed of natural or synthetic polymers. Another kind of ultrasound contrast agent includes suspensions of porous microparticles of polymers or other solids, which carry gas bubbles entrapped within the pores of the microparticles.
The present invention is particularly concerned with, although not limited to, the use of an ultrasound contrast agent (UCA) including an aqueous suspension of gas microbubbles, for exploiting a technique comprising the perfusion, destruction and monitoring of the replenishment of said UCA. Gas-filled microballoons may conveniently also be used for the present technique.
Microbubbles are typically defined as gas-filled microvesicles stabilized essentially by a layer of amphiphilic material. Aqueous suspensions of microbubbles are typically prepared by contacting powdered amphiphilic materials, e.g. freeze-dried preformed liposomes or freeze-dried or spray-dried phospholipid solutions, with air or other gas and then with aqueous carrier, and agitating to generate a microbubble suspension which must then be administered shortly after its preparation.
Examples of suitable aqueous suspensions of gas-filled microvesicles, in particular microbubbles and microballoons, and of the preparation thereof are disclosed, for instance, in the following patent applications: EP 0458745, WO 91/15244, EP 0554213, WO 94/09829 and WO 95/16467.
In 1998, investigators proposed to monitor the replenishment rate of a microbubble-based Ultrasound Contrast Agent (UCA), following destruction from an imaging plane by the ultrasound imaging instrument (Wei, K., Jayaweera, A. R., Firoozan, S., Linka, A., Skyba, D. M., and Kaul, S., “Quantification of Myocardial Blood Flow With Ultrasound-Induced Destruction of Microbubbles Administered as a Constant Venous Infusion,” Circulation, vol. 97 1998.). This possibility of destroying microbubbles locally essentially serves the purpose of providing a so-called “negative-bolus” of agent to the image plane, in an organ otherwise under an essentially constant perfusion of agent during the time of the measurement. Observation of the rate of reperfusion of UCA in the image plane, under continuous (i.e. so-called “realtime”) or intermittent (i.e. triggered) imaging, allowed an estimate of the organ perfusion, i.e. of the local flow-parameters.
This technique has been widely adopted. Extensive published literature has consistently reported using an optimal fit of the replenishment video or Doppler signal as a function of time, with an expression describing the dilution kinetics of an indicator in a single-compartment volume (in the form of a growing monoexponential function). See for example the following publications:    K. Wei, Detection and Quantification of Coronary Stenosis Severity With Myocardial Contrast Echocardiography, Progress in Cardiovascular Diseases, 44(2), 2001, 81-100: FIG. 8 of this reference shows a video intensity versus pulsing interval relation fitted to a monoexponential function.    Kevin Wei, Elizabeth Le, Jian-Ping Bin, Matthew Coggins, Jerrel Thorpe, Sanjiv Kaul. Quantification of Renal Blood Flow With Contrast-Enhanced Ultrasound. J. Am Col1 Cardiol, 2001; 37:1135-40: FIG. 2 of this reference shows the monoexponential relationship of video intensity versus pulsing interval(s).    Kharchakdjian, R., Burns, P. N., and Henkelman, M. Fractal Modeling of Microbubble Destruction-Reperfusion in Unresolved Vessels. IEEE Ultrasonics Symposium, 2001: This paper discusses the different types of reperfusion concentration-vs-time curves for different physiological flow conditions.    Rim, S.-J., Leong-Poi, H., Lindner, J. R, Couture, D., Ellegala, D., Masson, H. Durieux, M, Kasse, N. F. and Kaul S., Quantification of Cerebral Perfusion with Real-Time Contrast-Enhanced Ultrasound, Circulation, vol. 104, 2001, 2582-2587: FIGS. 2 and 3 of this reference show plots of acoustic intensity versus time, fitted by monoexponential functions, while the data recorded is described as proportional to agent concentration.    Schlosser et al, Feasibility of the Flash-Replenishment Concept in Renal Tissue: Which Parameters Affect the Assessment of the Contrast Replenishment?, Ultrasound in Med. & Biol., Vol. 27, pp 937-944, 2001: this article analyses contrast agent replenishment and also applies the nonlinear curve fitting using the monoexponential function introduced by Wei et al.    Murthy T H, Li P, Locvicchio E, Baisch C, Dairywala I, Armstrong W F, Vannan M. Real-Time Myocardial Blood Flow Imaging in Normal Human Beings with the use of Myocardial Contrast Echocardiography. J Am Soc Echocardiogr, 2001, 14(7):698-705: FIG. 7 of this reference shows that the video intensity versus time curve is fitted with the “1-phase exponential association equation”.    WO 02/102251 describes microbubble destruction/replenishment and shows in its FIG. 2b the monoexponential function of microvascular video intensity versus time, from which microvascular flow intensity is described as represented by the tangent to the initial slope of the monoexponential function. Its FIG. 2c shows the monoexponential function of video intensity versus pulsing interval (Intermittent mode).
The present inventors have observed that the prior heuristic approaches gave encouraging results because the echo signals in all echo-imaging instruments undergo heavy nonlinear compression (also called log-compression), before they are made available to the observer in the form of a video signal. Fitting the video data with the monoexponential function thus allowed to produce flow-estimates related to the actual local organ perfusion, which so far have been judged satisfactory.
The present inventors have however observed that the known approach is very sensitive to the user-selected instrument settings, such as receiver gain, log-compression, and so on. The parameters extracted are also specific to each instrument type, and thus cannot be compared between investigators using different equipment or settings. Furthermore, the perfusion parameters extracted from the state-of-the-art technique are only relative estimates, and are not suitable for an absolute quantitative evaluation of the flow parameters.